Antiviral compounds

ABSTRACT

The present invention relates to amino acid esters of pyrimidine and purine nucleosides containing an acyclic side chain, and their use in medical therapy, particularly the treatment of herpes virus infections. Also provided are pharmaceutical formulations and processes for the preparation of compounds according to the invention.

This is a divisional of copending application Ser. No. 07/712,058 filedJun. 7, 1991 which is a division of U.S. Ser. No. 07/452,543, filed Dec.18, 1989, now U.S. Pat. No. 5,043,339.

The present invention relates to novel antiviral esters of pyrimidineand purine nucleosides containing an acyclic side chain.

European Patent Specification 16785A describes and claims the antiviralpyrimidine nucleoside1-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]cytosine and itsphysiologically acceptable salts and esters. The parent compound hasbeen found to have particularly potent activity against cytomegalovirus(CMV) and Epstein-Barr virus (EBV).

The compound 9-[(2-hydroxy-1-hydroxymethylethoxy)methyl]guanine, whichhas the approved name ganciclovir, is described in UK PatentSpecification 2104070A which also describes generally thepharmaceutically acceptable salts and certain esters of ganciclovir.Ganciclovir has been found to have potent activity against viruses ofthe herpes family particularly herpes simplex and cytomegalovirus.Ganciclovir has however, low oral bioavailability and is typicallyadministered as a 1-hour intravenous infusion every 12 hours.

The 6-deoxy and 6-amino analogues of ganciclovir have also beendescribed in the literature, the former being described in UK PatentSpecification 2104070A and the latter is UK Patent Specification2130204A.

We have now found that amino acid esters of the compounds referred toabove surprisingly have advantageous bioavailability when administeredby the oral route, resulting in exceptionally high levels of the parentcompound in the body. This enables less drug to be administered whilestill providing equivalent drug levels of the parent compound in theplasma. Oral administration means patient compliance is considerablysimplified.

According to one feature of the present invention there is provided acompound of formula I: ##STR1## (wherein R and R¹ are independentlyselected from a hydrogen atom and an amino acid acyl residue providingat least one of R and R¹ represents an amino acid acyl residue and 3represents a group of formula ##STR2## in which R² represents a C₁₋₆straight chain. C₃₋₆ branched chain or C₃₋₆ cyclic alkoxy group, or ahydroxy or amino group or a hydrogen atom) and the physiologicallyacceptable salts thereof.

A group falling within formula (I) above is where R² represents ahydroxy or amino group or a hydrogen atom.

It will be appreciated that the compound of formula (I) in which 3represents a group of formula (B) wherein R² represents hydroxy is shownin the enol tautomeric form. The compound may also exist in its ketotautomeric form.

Among the above amino acid esters of formula (I) those of cytosine andganciclovir are particularly preferred by virtue of their especiallyimproved bioavailability in comparison with the parent compounds.

The amino acid acyl residue of the above compounds according to theinvention may be derived for example from naturally ocurring aminoacids, preferably neutral amino acids i.e. amino acids with one aminogroup and one carboxyl group. Examples of preferred amino acids includealiphatic acids, e.g., containing up to 6 carbon atoms such as glycine,alanine, valine and isoleucine. The amino acid esters according to theinvention includes the mono- and di-esters of the compound of formula(I). The amino acids may be D- L- and DL-amino acids, with the L-aminoacids being most preferred.

Examples of preferred compounds of formula (I) above include those ofExamples 1 to 6.

The above-mentioned physiologically acceptable salts are preferably acidaddition salts derived from an appropriate acid, e.g., hydrochloric,sulphuric, phosphoric, maleic, fumaric, citric, tartaric, lactic oracetic acid.

The above-defined amino acid esters of formula (I) and their salts whichare hereinafter referred to as the compounds according to the invention,are especially useful for the treatment of virus infections, especiallyherpes infections such as herpes simplex, varicella zoster, Epstein-Barrvirus (human herpes virus-6 infections), and particularlycytomegalovirus, in humans or non-human animals. Examples of clinicalconditions which are caused by such viruses include herpetic karatitis,herpetic encaphalitis, cold sores and genital infections (caused byherpes simplex), chicken pox and shingles (caused by varicella zoster)and CMV-pneumonia and retinitis, particularly in immunocompromisedpatients including renal and bone marrow transplant patients andpatients with Acquired Immune Deficiency Syndrome (AIDS). Epstein-Barrvirus (EVB) causes infectious mononucleosis, and is also suggested asthe causative agent of nasopharyngeal cancer, immunoblastic lymphoma,Burkitt's lymphoma and hairy leukoplakia.

According to further features of the present invention we provide

a) the compounds according to the invention for use in medical therapyparticularly for the treatment of viral infections, e.g., those referredto above:

b) the use of the compounds according to the invention for themanufacture of a medicament for the treatment of viral infections, e.g,those referred to above:

c) a method for the treatment of a herpes viral infection, especially acytomegalovirus infection, in a subject which comprises administering tothe subject an effective amount of a compound according to theinvention.

The compounds according to the invention may be administered for therapyby any route appropriate to the condition to be treated, suitable routesincluding oral, rectal, nasal, topical (including buccal andsublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous, intradermal, intrathecal, intraocular andepidural). It will be appreciated that the preferred route may vary withfor example the condition of the recipient.

For each of the above-indicated utilities and indications the amountrequired of the compound according to the invention will depend upon anumber of factors including the severity of the condition to be treatedand the identity of the recipient and will ultimately be at thediscretion of the attendant physician. In general however, for each ofthese utilities and indications, a suitable, effective dose will be inthe range 0.1 to 250 mg per kilogram bodyweight of recipient per day,preferably in the range 1 to 100 mg per kilogram bodyweight per day andmost preferably in the range 5 to 20 mg per kilogram bodyweight per day;an optimum dose is about 10 mg per kilogram bodyweight per day. Thedesired dose is preferably presented as two, three, four or moresub-doses administered at appropriate intervals throughout the day.These sub-doses may be administered in unit dosage forms, for example,containing 10 to 1000 mg, preferably 20 to 500 mg and most preferably100 to 400 mg of the compound according to the invention per unit dosageform.

The compounds of the invention may be administered for the treatment ofviral infections alone or in combination with other therapeutic agents,for example, with other antiviral agents such as9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) used to treat herpes viralinfections in particular HSV; with 3'-deoxy-3'-azidothymidine(zidovudine) or a 2',3'-dideoxynicleoside for example 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine or2',3'-dideoxyguanosine, used to treat retroviral infections inparticular Human Immunodeficiency Virus (HIV) infections, intarferonsparticularly α-interferon and soluble proteins such as CD4, or any otheragents such as analagesics or antipyretics which when in combinationwith a compound of the invention provide a beneficial therapeuticeffect.

While it is possible for the active ingredients to be administered aloneit is preferable to present them as pharmaceutical formulations. Theformulations, both for veserinary and for human use, of the presentinvention comprise at least one compound according to the invention(also referred to hereafter as "the active ingredient"), together withone or more acceptable carriers therefor and optionally othertherapeutic ingredients. The carrier(s) must be "acceptable" in thesense of being compactible with the other ingredients of the formulationand not deleterious to the receipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intrathecal,intraocular and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. Such methods includethe step of bringing into association the active ingredient with thecarrier which constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers of finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

For infections of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient in an amount of, for example,0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to10% w/w. When formulated in an ointment, the active ingredients may beemployed with either paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream withan oil-in-water cream base.

If desired, the aqueous phase of the cream base may include, forexample, at least 30% w/w of polyhydric alcohol, i.e., an alcohol havingtwo or more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulphoxide and related analogues.

Formulations suitable for topical administration to the eye also includedye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerine, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for nasal administration wherein the carrier is asolid include a coarse powder having a particle size for example in therange 20 to 500 microns which is administered in the manner in whichsnuff is taken, i.e., by rapid inhalation through the nasal passage froma container of powder held close up to the nose. Suitable formulationswherein the carrier is a liquid, for administration as for example anasal spray or as nasal drops, include aqueous or oily solutions of theactive ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents and liposomes or other microparticulate systems which aredesigned to target the compound to blood components or one or moreorgans. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described. Formulations for intramuscularadministration are particularly preferred.

Preferred unit dosage formulations are those containing a daily dose orunit daily sub-dose, as herein above recited, or an appropriate fractionthereof, of an active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The present invention further provides veterinary compositionscomprising at least one active ingredient as above defined together witha veterinary carrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

For oral administration the compositions can be in the form of a tablet,granule, drench, paste, cachet, capsule or feed supplement. Granules maybe made by the well known techniques of wet granulation, precompressionor slugging. They can be administered to animals in an inert liquidvehicle so as to form a drench, or in a suspension with water or oilbase. Preferably further accessory ingredients such as a dispersingagent are includes. These formulations preferably contain from 15 to 85%of the active ingredient.

The compounds according to the invention may be prepared in conventionalmanner, e.g. by a process as described below.

Thus, according to a further feature of the present invention we providea process for the preparation of the compounds according to theinvention which comprises reacting the compound of formula (II) ##STR3##(wherein B is as hereinbefore defined) with an optionally protectedamino acid or functional equivalent thereof and optionally effecting oneor more of the following conversions:

i) removal of any protecting groups;

ii) where the resulting product is a compound of formula (I), conversionof the said compound into a physiologically acceptable salt thereof; and

iii) where the resulting product is a physiologically acceptable salt ofa compound of formula (I), conversion of the said salt into the parentcompound.

In the above process, the reaction may be carried out in a conventionalmanner, for example in a solvent such as pyridine, dimethylformamideetc., in the presence of a coupling agent such as N,N'-dicyclohexylcarbodiimide, optionally in the presence of a catalytic basesuch as 4-dimethylaminopyridine. The water formed during the reactionmay, if desired, by removed in conventional manner, for example bydistillation or by the addition of a water-binding substance.Subsequently, the ester obtained as reaction product may be isolated inconventional manner.

As an alternative to the use of the amino acid per se, a functionalequivalent of the acid may be employed, e.g., an acid halide such as theacid chloride, or an acid anhydride.

In order to avoid undesirable side-reactions, it may be advantageous touse an amino-protected derivative, examples of preferredamino-protecting groups including acyl, e.g., C₁₋₄ alkanoyl such asacetyl; arylalkyloxycarbonyl, e.g., benzyloxycarbonyl; oramino-precursor groups such as azido groups. It is particularlypreferred to employ an amino acid protected by a benzyloxycarbonylgroup. Such benzyloxycarbonyl protected compounds are commerciallyavailable, e.g., from Sigma Chemical Co., USA, or may be prepared bytreating the appropriate amino acid with carbobenzoxy chloride inalkaline solution.

The optional conversions i), ii) and iii) may be effected in aconventional manner. Thus, for example, removal of protecting groups inconversion i) may be effected by hydrogenolysis or as appropriate. Withregard to removal of protecting groups on the amino acid acyl radicals,hydrogenolysis, e.g., of arylalkyloxycarbonyl protecting groups, andconversion of azido group, e.g., by catalytic hydrogenation, e.g., usinga palladium catalyst, are preferred.

The conversion of an amino acid ester into a physiologically acceptablesalt may be effected in conventional manner, e.g., by treatment of thecompound with an appropriate acid to form an acid addition salt.

Similarly, conversion of a salt into the parent amino acid ester may beeffected in conventional manner for example, by treatment with astoichiometric amount of an ion exchange resin (basic form), filtrationto remove the resin and lyophilisation of the reslting solution.

The following Examples illustrate the present invention.

EXAMPLE 1

a)2-((4-(N-((Benzyloxy)carbonyl)-L-isoleucinamido)-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(N-((benzyloxy)-carbonyl)-L-isoleucinate) and2-((4-Amino-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(N-((benzyloxy)-carbonyl)-L-isoleucinate)

A suspension of 2 g of4-amino-1-((2-hydroxy-1-(hydroxymethyl)-ethoxy)methyl)-2(1H)-pyrimidinonein 40 mL dry dimethylformamide (DMF) was warmed to 60° C. to give acolorless solution. 7.4 g of CBz-L-isoleucine, 567 mg ofdimethylaminopyridine (DMAP) and 5.75 g of dicyclohexylcarbodiimide(DCC) were successively added. A white precipitate was observed after 15min. The mixture was allowed to stir at room temperature for 4 days. Theresulting suspension was filtered and the filtrate was dried (MgSO₄),filtered and evaporated in vacuo to a light yellow oil. The oil waspurified by flash chromatography on silica gel, eluting with 4:1EtOAC-Hex to afford the triacylated derivative, 783 mg (9%), as a whitefoam. Elution with 5:1 EtOAc-MeOH afforded the diacylated derivative,2.93 g (44%) as a white foam.

b) 2-((4-amino-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(L-isoleucinate)

To a cool mixture of 2.54 g of2-((4-amino-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-isoleucinate) and 5.16 g of 10% palladiumcatalyst in acetic acid was slowly added 6.4 mL of 1,4-cyclohexadiene.The mixture was allowed to stir at room temperature for 3 h. Thereaction mixture was filtered through a pad of Celite, the filtrate wasthen concentrated and dried in the lyophiliser for 3 d. The residue wasscraped off, affording 2.079 g (82%, as the acetic acid salt) of a beigefoam. The elemental analysis (showing 2M of acetic acid and 1M ofwater), UV, ¹ H. ¹³ C-NMR spectra were consistent with the titlestructure.

EXAMPLE 2

a)2-((4-(N-(benzyloxy)carbonyl)-L-valinamido)-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-valinate) and2-((4-amino-1,2-dihydro-2-oxo-1-pyrimidinyl)-methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-valinate)

A suspension of 2 of4-amino-1-((2-hydroxy-1-(hydroxymethyl)-ethoxy)methyl)-2(1H)-pyrimidinonein 40 mL dry dimethylformamide (DMF) was slightly warmed until a clearsolution remained. 5.84 g of CBz-L-valine, 567 mg ofdimethylaminopyridine (DMAP) and 4.79 g of dicylohexylcarbodiimide (DCC)was successively added. A white precipitate was observed after 15 min.The mixture was stirred at room temperature for 4 h. The resultingsuspension was filtered and the filtrate distributed between water andCH₂ Cl₂. The organic fraction was dried (MgSO₄), filtered and evaporatedin vacuo to a yellow oil. The oil was purified by flash chromatographyof silica gel. Eluting with 2% methanol in dichloromethane afforded thetriacylated derivative, 1.12 g (13%) as a white foam. Eluting with 4%methanol in dichloromethane afforded the N,O-diacylated derivative, 113mg (2%). Eluting with 10% methanol in dichloromethane afforded theO,O-diacylated derivative, 3.44 g (54%) as a white foam.

b) 2-((4-amino-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(L-valinate)

To a cool mixture of 3.44 g of2-((4-amino-1,2-dihydro-2-oxo-1-pyrimidinyl)methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-valinate) and 7 g of 10% palladiumcatalyst in acetic acid was slowly added 8.65 mL of 1,4-cyclohexadiene.The mixture was allowed to stir at room temperature for 18 h. Thereaction mixture was filtered through a pad of Celite. The filtrate wasthen concentrated and dried in the lyophilizer for 48 h. The resultingbeige foam was scraped off affording 2.38 g (68% as the acetic acidsalt) of the title compound. The elemental analysis (showing 2 M ofacetic acid and 1 M of water) UV, ¹ H, ¹³ C-NMR spectra were consistentwith the title structure.

EXAMPLE 32-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(L-isoleucinate)

a) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-isoleucinate)

1,3-Dicyclohexylcarbodiimide (6.2 g, 0.03 mol, Aldrich) was added to astirring suspension of N-benzyloxycarbonyl-L-isoleucine (7.96 g, 0.03mol, Sigma), 9-((2-hydroxy-1-hydroxymethylethoxy)methyl) guanine (2.55g, 0.01 mol), and 4-dimethylaminopyridine (0.4 g, 0.003 mol, Aldrich) inN,N-dimethylformamide (150 ml). The mixture was stirred at roomtemperature for four days then collected by filtration and washed withdimethylformamide (20 ml). The filtrate, including wash, was evaporatedunder vacuum to a slurry. A solution of this residue in dichloromethane(100 ml) was filtered, treated with silica gel (50 g, E, Mark, 230-400mesh), and concentrated under vacuum. The residue was added to a silicagel column and subjected to flash chromatography with gradient elutionstarting with 100% dichloromethane and gradually increasing to 7%methanol. The product-containing fractions were concentrated undervacuum, dissolved in dichloromethane (50 ml) and washed with water(5×100 ml) to remove residual dimethylformamide. The organic layer wasdried over magnesium sulphate, filtered, and evaporated under vacuum togive 4.3 g (57%) of a white solid, Mp. -60° C.(dec.):TLC(silica gel, CH₂Cl₂ :MeOH/9:1)R_(f) -0.63:H-NMR(DMSO-d₆)10.57(br s.1H), 7.78(s, 1H),7.63(d,d,2H), 7.33(m,10H), 6.41(br s,2H), 5.41(s,2H), 5.01(s,4H)m3.87-4.23(m,7H), 1.68(m,2H), 1.20(m,4H), 0.755(m,12H): HPLC(VersapackC-18; 70% MeOH(aq.)/0.1%TFA)k'-1.76;MS(cl)m/z 750 (100%.M+H); UV max(MeOH)238.8 nm(22,300). Anal. Calcd. for C₃₇ H₄₇ N₇ O₁₀ : C,59.27;H,6.32: N,13.08. Found: C,59.18: H,6.38; N,13.02.

b) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)-1,3-propanediylbis(L-isoleucinate)

A solution of the product of stage a) (2.0 g, 2.66 mmol) and 10%palladium on carbon (250 mg, Aldrich) in glacial acetic acid (250 ml)was shaken on a Parr Hydrogenator under 30 to 35 psi H₂ for 5 hours.Reaction progress was monitored by TLC (silica gel, CH₂ Cl₂ :MeOH/9:1).The catalyst was removed by filtration (Millipore Prefilter). Dilutionof the filtrate with either caused the product to separate as an oil.After 14 hours at 5° C. the supernatant was decanted from a colourlessgum, which was dried under high vacuum to a white foam. The foam wasdissolved in water (20 ml), filtered, and freeze-dried to a white foam:yield, 1.3 g(79%). H-NMR (DMSO-d₆) 7.79 (3.1H), 6.49 (br s,2H), 5.41(s,2H), 3.9-4.2 (m,4H), 3.10 (d,1H), 3.06 (d,1H), 1.9 (HOAc), 1.5(m,1H), 1.34 (m,4H), 1.1 (m,4H); 0.73-0.78 (m,12H), ¹³ C-NMR (DMSO-d₆)156.80, 153.96, 151.30, 137.47, 116.30, 73.41, 70.78, 62.88, 62.57,58.15, 24.18, 21.28, 15.33, 15.28, 11.33; HPLC (Versapack C-18: 30% MeOH(aq.)/0.1% TFA) 1 peak, k'-1.22; UV max (H₂ O) 251 nm (12,000), MS(positive ion/Cl) m/z 482. Anal. Calcd. for C₂₁ H₃₅ N₇ O₆.² HOAc:1H₂ O:C,48.46; H,7.32: N,15.82. Found: C,48.52: H,7.3; N,15.75.

EXAMPLE 42((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(2-aminoacetate)

a) 2-((2-Amino-1,6-dihydro-6oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(N-((benxyloxy)carbonyl)glycinate)

9-((2-Hydroxy-1-hydroxymethylethoxy)methyl)guanine (2.55 g, 0.01 mol),N-benzyloxycarbonyl-glycine (6.28 g, 0.03 mol, Aldrich), and1,3-dicyclohexylcarbodiimide (6.2 g, 0.03 mol, Aldrich) were added toN,N-dimethylformamide (100 ml) and stirred at room temperature for twodays. The white precipitate was colected by filtration and washed withdimethylformamide (50 ml). The filtrate, including wash, was evaporatedunder vacuum to a slurry. A solution of the residue in dichloromethane(100 ml) was filtered and washed with brine solution (3×125 ml). Theorganic layer was dried over magnesium sulphate, filtered and evaporatedunder vacuum to a solid. A solution of the solid in dichloromethane (20ml) was purified on a silica gel column (E.Merk, 230-400 mesh) by flashchromatography and gradient elution, starting with 100% dichloromethaneand increasing by 2% increments to 8% methanol. The product fractionswere evaporated to dryness under vacuum to give 5.20 g (80%) of a whitepowder, M.P. -120° C. (dec.):TLC (silica gel, CH₂ Cl₂ :MeOH/9:1) R_(f)-0.38; H-NMR(DMSO-d₆)10.60(s,LH), 7.30 (s,1H), 7.65 (t,2H), 7.32(m,10H), 6.46 (br s.2H), 5.40 (s,2H), 5.02 (s,4H), 4.05 (m,5H), 3.68(d,4H); HPLC Varsapack C-18; 30% MeOH (aq.)/0.1% TFA)k'-0.89; UV max(MeOH) 254.4 nm (16.100). Anal. Calcd. for C₂₉ H₃₁ N₇ O₁₀.O.5H₂ O:C,53.87; H,4.99; N,15.16. Found: C,53.99; H,5.02; N,15.13.

b) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)-1,3-propanediylbis(2-aminoacetate)

A solution of the product of stage a) (2.0 g. 3 mmol) and 10% palladiumon carbon (250 mg, Aldrich) in glacial acetic acid (250 ml) was shakenon a Parr Hydrogenator under 30 to 35 psi H₁ for 7 hours 19 minutes,monitored by TLC on silica gel using 10% methanol in methylene chloride.The catalyst was removed by filtration (Millipore Prefilter), thefiltrate concentrated to 10 ml by vacuum, and then freeze-dried. Thesolid was dissolved in water, filtered, and freeze-dried again to give1.94 g (100%) of a white solid. H-NMR (DMSO-d₆) 7.79 (t,1H), 6.62 (brs,2H), 5.40 (s,2H), 4.02 (m,4-5H), 3.19 (s,4H) ¹³ C-NMR (DMSO-d₆)169.40, 166.04, 156.83, 153.97, 153.88, 151.31, 151.22, 137.51, 116.28,79.92, 76.52, 73.40, 71.39, 71.20, 71.07, 63.77, 63.02, 60.76, 60.18,44.25, 42.31, 42.18; HPLC (Versapack C-18; 20% MeOH (aq.)/0.1% TFA) 2peaks, k'-0.06, 0.18; UV max (H₂ O) 252 nm (12,100), 263.6 nm (8,790).Anal. Calcd. for C₁₃ H₁₉ N₇ O₆ 4HOAc 0.5H₂ O: C,40.78; H,5.87; N,15.85.Found: C,40.99: H,5.93: N, 15.70.

EXAMPLE 52-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy-1,3-propanediylbis(L-valinate)

a) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-valinate)

A solution of 22.5 g(0.09M) of N-benzyloxycarbonyl chloride (SigmaChemical Co.) L.valine, 18.6 g (0.09 mol) ofN,N-dicyclohexylcarbodiimide and 1.2 g (0.01 mol) of4-dimethylaminopyridine in 100 mL of dimethylformamide was stirred undernitrogen for 10 minutes. After the addition of 20 mL more ofdimethylformamide and 7.65 g (0.03 mol) of9-(1,3-dihydroxypropoxymethyl)guanine, the mixture was stirred for 18hours at ambient temperature.

The suspension was filtered, washing the precipitate withdichloromethane and the combined filtrate and washings were evaporatedin vacuo. The residual yellow oil was dissolved in methanol and absorbedon silica gel. The mixture was evaporated in vacuo and the powderyresidue added to a column prepared for flash chromatography. The columnwas eluted first with 2% methanol in dichloromethane to remove animpurity and the desired product was then eluted off with 5% methanol indichloromethane. Evaporation of this eluate gave 14.3 g (66%) of2-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediyl bis(N-((benzyloxy)carbonyl)-L-valinate), which gave a satisfactoryelemental analysis, ¹ H HMR and ¹³ C spectra.

b) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(L-valinate)

A mixture of 0.722 g (1.0 mmol) of 2-((2-amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-valinate) and 300 mg of 10% palladium oncarbon in 10 mL of acetic acid was shaken in a Parr apparatus at ambienttemperature at an initial pressure of 50 psi for 18 hours. The mixturewas filtered through a pad of celite, washing the pad with acetic acid.The filtrate was evaporated at room temperature under pump vacuum givinga syrup (822 mg) which was dried at 100° C. with 1.0 mm pressure. Theresulting glass turned to a solid on scraping with a spatula and was thedesired2-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(L-valinate). The compound gave satisfactory ¹ H NMR, ¹³ C, UV andMass spectra. It analyzed for 2 moles of acetic acid and 0.05 moles ofwater.

EXAMPLE 62-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)-1,3-propanediylbis(L-alaninate)

a) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy-1,3-propanediylbis(N-((benzyloxy)carbonyl)-L-alaninate)

A mixture of 13.4 g (0.06 mol) N-benzyloxycarbonyl-L-alanine (Sigmachemical company), 12.4 g (0.06 mol) of N,N-dicyclohexylcarbodiimide,0.8 g (0.006 mole) of 4-dimethylaminopyridine and 5.1 g (0.02 mol) of9-(1,3-dihydroxypropoxymethyl)guanine in 640 mL of dimethylformamide wasstirred under nitrogen 18 hours at ambient temperature.

The suspension was filtered, washing the precipitate withdichloromethane and the combined filtrate and washings were evaporatedin vacuo. The residual yellow oil was dissolved in methanol and absorbedon silica gel. The mixture was evaporated in vacuo and the powderyresidue added to a column prepared for flash chromatography. The columnwas eluted first with 1:1 ethyl acetate and ether, then with acetone, toremove impurities. The desired product was then eluted off with 5% and10% methanol in dichloromethane. Evaporation of these eluates gave 7.649g (57%) of2-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3propanediylbis(N-((benzyloxy)carbonyl)-L-alaninate), which gave satisfactory ¹ HNMR and ¹³ C spectra.

b) 2-((2-Amino-1,6-dihydro-6-oxo-9H-purin-9yl)methoxy)-1.3-propanediylbis(L-alaninate)

A mixture of 1.0 g (1.5 mmol) of2-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-1,3-propanediylbis(N-((benxyloxy) carbonyl)-L-alaninate) and 500 mg of 10% palladium oncarbon in 10 mL of acetic acid was shaken in Parr apparatus at ambienttemperature at an initial pressure of 42 psi for 18 hours. The mixturewas filtered through a pad of celite, washing the pad with acetic acid.The filtrate was lyophilized giving the desired2-((2-amino-1,6-dihydro-6-oxy-9H-purin-9-yl)methoxy)-1,3-propanediylbis(L-alaninate) as a syrup (0.98 g). Analysis by ¹ H NMR. ¹³ C NMR,HPLC(C-18 reverse phase in 15% of 0.1% trifluoroaceticacid-acetonitrile/0.1% trifluoroacetic acid-water) and Mass spectraproved that the compound was a mixture of the O-monoesterified andO,O-diesterified in the ratio of 1:9.

The following Examples, 7 to 9, illustrate pharmaceutical formulationsaccording to the invention where the active ingredient is a compoundaccording to the invention.

    ______________________________________                                        Example 7               Tablet                                                ______________________________________                                        Active compound         200 mg                                                Lactose                 235 mg                                                Starch                   50 mg                                                Polyvinylpyrrolidone     50 mg                                                Magnesium stearate       5 mg                                                 ______________________________________                                    

Mix the active compound with the lactose and starch and wet granulatewith a solution of the polyvinylpyrrolidone. Dry, sift, blend thegranules with magnesium stearate and compress.

    ______________________________________                                        Example 8              Capsule                                                ______________________________________                                        Active compound        200 mg                                                 Lactose                184 mg                                                 Sodium starch glycollate                                                                              8 mg                                                  Polyvinylpyrrolidone    6 mg                                                  Magnesium stearate      2 mg                                                  ______________________________________                                    

Mix the active compound with the lactose and sodium starch glycollateand wet granulate with a solution of the polyvinylpyrrolidone. Dry,sift, blend the granules with the magnesium stearate and fill into hardgelatin capsules.

    ______________________________________                                        Example 9            Intravenous Injections                                   ______________________________________                                        A)    Active compound    200 mg                                                     Sodium hydroxide solution                                                                        q.s. to pH 7.0 to 7.5                                      Water for injections to                                                                          5.0 ml                                               ______________________________________                                    

Dissolve the active compound in part of the water for injections. Adjustthe pH with the sodium hydroxide solution and make up to volume withadditional water for injections. Under aseptic conditions, sterilise thesolution by filtration, fill into sterile ampoules and seal theampoules.

    ______________________________________                                        B)    Active compound    100 mg                                                     Sodium hydroxide solution                                                                        q.s. to pH 7.0 to 7.5                                      Mannitol           125 mg                                                     Water for injections to                                                                          2.5 ml                                               ______________________________________                                    

Dissolve the active compound and mannitol in part of the water forinjections. Adjust the pH with the sodium hydroxide solution and make upto volume with additional water for injections. Under asepticconditions, sterilise the solution by filtration, fill into sterilevials and remove the water by freeze-drying. Seal the vials under anatmosphere of nitrogen and close with a sterile stopper and aluminiumcollar.

I claim:
 1. A compound of formula (I): ##STR4## wherein R and R¹ areL-valyl and B represents a group of formula ##STR5## or aphysiologically acceptable salt thereof.
 2. A method of treatment ofherpes viral infection in a subject which comprises administering to thesubject an effective antiherpes viral infection treatment amount of thecompound of formula (I) as defined in claim 1 or a physiologicallyacceptable salt thereof.
 3. A method according to claim 2, in which theinfection is a cytomegalovirus infection and the amount is an effectiveanticytomegalovirus infection treatment amount.
 4. A pharmaceuticalformulation comprising as active ingredient a compound of formula (I) asclaimed in claim 1 or a physiologically acceptable salt thereof togetherwith at least one pharmaceutically acceptable carrier therefor.
 5. Apharmaceutical formulation as claimed in claim 4, adapted for oral orparenteral administraion.
 6. A pharmaceutical formulation as claimed inclaim 4, in the form of a tablet or capsule.
 7. A physiologicallyacceptable acid addition salt of the compound of formula (I) ##STR6##wherein B represents a group of the formula ##STR7## and R and R¹ areL-valine.
 8. The physiologically acceptable acid addition salt of claim7 derived from fumaric acid.
 9. A method of treating a herpes viralinfection in a subject which comprises administering to said subject aneffective antiherpes treatment amount of a physiologically acceptableacid addition salt of the compound of formula (I) ##STR8## wherein Brepresents a group of the formula ##STR9## and R and R¹ are L-valyl. 10.The method of claim 9 in which the acid addition salt administered isderived from fumaric acid.
 11. The method of claim 9 in which the herpesviral infection is a cytomegalovirus infection and the amountadministered is an effective anticytomegalovirus infection treatmentamount.
 12. The method of claim 9 in which the salt administered isderived from fumaric acid, and in which the herpes viral infection is acytomegalovirus infection and the amount administered is an effectiveanticytomegalovirus infection treatment amount.
 13. The method of claims9, 10, 11 and 12 in which the salt is administered orally.
 14. Apharmaceutical composition comprising as the active ingredient the saltof claim 7 together with at least one pharmaceutically acceptablecarrier therefore.
 15. The composition according to claim 14 in whichthe salt is derived from fumaric acid.